Antibodies to human resistin

ABSTRACT

The present invention relates to the field of pulmonary, cardiac and inflammatory disorders. More specifically, the present invention provides methods and compositions for treating disorders associated with Resistin. In a specific embodiment, the present invention provides an antibody that binds human Resistin.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.61/835,855, filed Jun. 17, 2013, which is incorporated herein byreference in its entirety.

STATEMENT OF GOVERNMENTAL INTEREST

This invention was made with government support under grant no.P50HL107182 awarded by the National Institutes of Health. The governmenthas certain rights in the invention.

FIELD OF THE INVENTION

The present invention relates to the field of pulmonary, cardiac andinflammatory disorders. More specifically, the present inventionprovides methods and compositions for treating disorders associated withResistin.

INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY

This application contains a sequence listing. It has been submittedelectronically via EFS-Web as an ASCII text file entitled“P12269-02_Sequence_Listing.txt.” The sequence listing is 291,637 bytesin size, and was created on Jun. 17, 2014. It is hereby incorporated byreference in its entirety.

BACKGROUND OF THE INVENTION

Resistin and Resistin-Like Molecule-Beta (RELM-beta) are the humananalogs of a recently identified family of secreted proteins containinga conserved cysteine-rich C-terminus. The RELM family consists ofResistin (also called FIZZ3), RELM-alpha (FIZZ1), RELM-beta (FIZZ2) andRELM-gamma (FIZZ4). In human there are two isoforms, Resistin andRELM-Beta. The RELM family of proteins has been implicated in severalhuman disease states including, but not limited to, pulmonaryhypertension, cardiac hypertrophy and failure, asthma, lunginflammation, sepsis, acute lung injury, respiratory distress syndrome,pulmonary fibrosis, scleroderma, arteriosclerosis, chronic obstructivelung disease/emphysema, normal and abnormal wound healing, cancer, cellproliferation, stem cell growth and differentiation, diabeticretinopathy, and insulin resistance. There is a continuing need in theart to provide therapies for diseases, disorder and conditions mediatedby the RELM family of proteins.

SUMMARY OF THE INVENTION

The present invention is based, at least in part, on the development ofantibodies to Resistin. Thus, in particular embodiments, the presentinvention provides isolated antibodies that bind human Resistin. Suchantibodies may also be cross-reactive with RELMB. In particularembodiments, the antibodies are recombinant or otherwise non-naturallyoccurring antibodies. In other embodiments, the present inventionprovides nucleotide sequences that encode an antibody that binds humanResistin. The present invention further provides amino acid sequencesthat encode an antibody that binds human Resistin. The antibody can be asingle chain variable fragment (scFv), a dimeric scFv, a Fab, a Fab′, aF(ab′)2 fragment or a full length antibody.

In specific embodiments, the antibody comprises a variable heavy chaincomprising SEQ ID NO:3, SEQ ID NO:13, SEQ ID NO:23, SEQ ID NO:33, SEQ IDNO:43, SEQ ID NO:53, SEQ ID NO:63, SEQ ID NO:73, SEQ ID NO:83, SEQ IDNO:93, SEQ ID NO:103, SEQ ID NO:113, SEQ ID NO:123, SEQ ID NO:133, SEQID NO:143, SEQ ID NO:153, SEQ ID NO:163, or fragments thereof. In otherembodiments, the antibody comprises a variable heavy chain that is atleast 90% identical to SEQ ID NO:3, SEQ ID NO:13, SEQ ID NO:23, SEQ IDNO:33, SEQ ID NO:43, SEQ ID NO:53, SEQ ID NO:63, SEQ ID NO:73, SEQ IDNO:83, SEQ ID NO:93, SEQ ID NO:103, SEQ ID NO:113, SEQ ID NO:123, SEQ IDNO:133, SEQ ID NO:143, SEQ ID NO:153, SEQ ID NO:163, or fragmentsthereof.

In certain embodiments, the antibody comprises a light chain comprisingSEQ ID NO:7, SEQ ID NO:17, SEQ ID NO:27, SEQ ID NO:37, SEQ ID NO:47, SEQID NO:57, SEQ ID NO:67, SEQ ID NO:77, SEQ ID NO:87, SEQ ID NO:97, SEQ IDNO:107, SEQ ID NO:117, SEQ ID NO:127, SEQ ID NO:137, SEQ ID NO:147, SEQID NO:157, SEQ ID NO:167, or fragments thereof. Alternatively, theantibody comprises a light chain that is at least 90% identical to SEQID NO:7, SEQ ID NO:17, SEQ ID NO:27, SEQ ID NO:37, SEQ ID NO:47, SEQ IDNO:57, SEQ ID NO:67, SEQ ID NO:77, SEQ ID NO:87, SEQ ID NO:97, SEQ IDNO:107, SEQ ID NO:117, SEQ ID NO:127, SEQ ID NO:137, SEQ ID NO:147, SEQID NO:157, SEQ ID NO:167, or fragments thereof.

The present invention also provides antibodies in which the variabledomain of the heavy chain comprises one or more complementaritydetermining regions (CDRs) selected from the group consisting of SEQ IDNO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:14, SEQ ID NO:15, SEQ IDNO:16, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:34, SEQ IDNO:35, SEQ ID NO:36, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ IDNO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:64, SEQ ID NO:65, SEQ IDNO:66, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:4, SEQ IDNO:85, SEQ ID NO:86, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ IDNO:104, SEQ ID NO:105, SEQ ID NO:106, SEQ ID NO:114, SEQ ID NO:115, SEQID NO:116, SEQ ID NO:124, SEQ ID NO:125, SEQ ID NO:126, SEQ ID NO:134,SEQ ID NO:135, SEQ ID NO:136, SEQ ID NO:144, SEQ ID NO:145, SEQ IDNO:146, SEQ ID NO:154, SEQ ID NO:155, SEQ ID NO:156, SEQ ID NO:164, SEQID NO:165, SEQ ID NO:166, and fragments thereof. In other embodiments,the variable domain of the heavy chain comprises one or morecomplementarity determining regions (CDRs) that are at least 90%identical to a CDR selected from the group consisting of SEQ ID NO:4,SEQ ID NO:15, SEQ ID NO:6, SEQ ID NO:14, SEQ ID NO:5, SEQ ID NO:16, SEQID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:34, SEQ ID NO:35, SEQ IDNO:36, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:54, SEQ IDNO:55, SEQ ID NO:56, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ IDNO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:4, SEQ ID NO:85, SEQ IDNO:86, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:104, SEQ IDNO:105, SEQ ID NO:106, SEQ ID NO:114, SEQ ID NO:115, SEQ ID NO:116, SEQID NO:124, SEQ ID NO:125, SEQ ID NO:126, SEQ ID NO:134, SEQ ID NO:135,SEQ ID NO:136, SEQ ID NO:144, SEQ ID NO:145, SEQ ID NO:146, SEQ IDNO:154, SEQ ID NO:155, SEQ ID NO:156, SEQ ID NO:164, SEQ ID NO:165, SEQID NO:166, and fragments thereof.

The present invention also provides antibodies in which the variabledomain of the light chain comprises one or more CDRs selected from thegroup consisting of SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ IDNO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:28, SEQ ID NO:29, SEQ IDNO:30, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:48, SEQ IDNO:49, SEQ ID NO:50, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ IDNO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:78, SEQ ID NO:79, SEQ IDNO:80, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:98, SEQ IDNO:99, SEQ ID NO:100, SEQ ID NO:108, SEQ ID NO:109, SEQ ID NO:110, SEQID NO:118, SEQ ID NO:119, SEQ ID NO:120, SEQ ID NO:128, SEQ ID NO:129,SEQ ID NO:130, SEQ ID NO:138, SEQ ID NO:139, SEQ ID NO:140, SEQ IDNO:148, SEQ ID NO:149, SEQ ID NO:150, SEQ ID NO:158, SEQ ID NO:159, SEQID NO:160, SEQ ID NO:168, SEQ ID NO:169, SEQ ID NO:170, and fragmentsthereof. In alternative embodiments, the variable domain of the lightchain comprises one or more CDRs that are at least 90% identical to aCDR selected from the group consisting of SEQ ID NO:8, SEQ ID NO:9, SEQID NO:10, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:28, SEQ IDNO:29, SEQ ID NO:30, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ IDNO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:58, SEQ ID NO:59, SEQ IDNO:60, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:78, SEQ IDNO:79, SEQ ID NO:80, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ IDNO:98, SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:108, SEQ ID NO:109, SEQ IDNO:110, SEQ ID NO:118, SEQ ID NO:119, SEQ ID NO:120, SEQ ID NO:128, SEQID NO:129, SEQ ID NO:130, SEQ ID NO:138, SEQ ID NO:139, SEQ ID NO:140,SEQ ID NO:148, SEQ ID NO:149, SEQ ID NO:150, SEQ ID NO:158, SEQ IDNO:159, SEQ ID NO:160, SEQ ID NO:168, SEQ ID NO:169, SEQ ID NO:170, andfragments thereof.

In specific embodiments, the present invention provides an scfv thatbinds human Resistin, wherein the scfv is encoded by SEQ ID NO:1, SEQ IDNO:11, SEQ ID NO:21, SEQ ID NO:31, SEQ ID NO:41, SEQ ID NO:51, SEQ IDNO:61, SEQ ID NO:71, SEQ ID NO:81, SEQ ID NO:91, SEQ ID NO:101, SEQ IDNO:111, SEQ ID NO:121, SEQ ID NO:131, SEQ ID NO:141, SEQ ID NO:151, SEQID NO:161 or fragments thereof. In other embodiments, the scfv isencoded by nucleotide sequence that is at least 90% identical to SEQ IDNO:1, SEQ ID NO:11, SEQ ID NO:21, SEQ ID NO:31, SEQ ID NO:41, SEQ IDNO:51, SEQ ID NO:61, SEQ ID NO:71, SEQ ID NO:81, SEQ ID NO:91, SEQ IDNO:101, SEQ ID NO:111, SEQ ID NO:121, SEQ ID NO:131, SEQ ID NO:141, SEQID NO:151, SEQ ID NO:161 or fragments thereof.

The present invention also provides an scfv that binds human Resistin,wherein the scfv comprises SEQ ID NO:2, SEQ ID NO:12, SEQ ID NO:22, SEQID NO:32, SEQ ID NO:42, SEQ ID NO:52, SEQ ID NO:62, SEQ ID NO:72, SEQ IDNO:82, SEQ ID NO:92, SEQ ID NO:102, SEQ ID NO:112, SEQ ID NO:122, SEQ IDNO:132, SEQ ID NO:142, SEQ ID NO:152, SEQ ID NO:162 or fragmentsthereof. In alternative embodiments, the scfv comprises an amino acidsequence that is at least 90% identical to SEQ ID NO:2, SEQ ID NO:12,SEQ ID NO:22, SEQ ID NO:32, SEQ ID NO:42, SEQ ID NO:52, SEQ ID NO:62,SEQ ID NO:72, SEQ ID NO:82, SEQ ID NO:92, SEQ ID NO:102, SEQ ID NO:112,SEQ ID NO:122, SEQ ID NO:132, SEQ ID NO:142, SEQ ID NO:152, SEQ IDNO:162 or fragments thereof.

The antibodies of the present invention can further comprise a constantdomain comprising SEQ ID NO:172, SEQ ID NO:174 or a fragment thereof. Inother embodiments, the antibodies can further comprise a constant domainthat is at least 90% identical to SEQ ID NO:172, SEQ ID NO:174 or afragment thereof.

In specific embodiments, the present invention also provides a Resistinantibody comprising a heavy chain selected from the group consisting ofSEQ ID NO:176, SEQ ID NO:180, SEQ ID NO:184, SEQ ID NO:188, SEQ IDNO:192, SEQ ID NO:196, SEQ ID NO:200, SEQ ID NO:204, SEQ ID NO:208, SEQID NO:212, SEQ ID NO:216, SEQ ID NO:220, SEQ ID NO:224, SEQ ID NO:228,SEQ ID NO:232, SEQ ID NO:236, and SEQ ID NO:240. In additionalembodiments, a Resistin antibody comprises a light chain selected fromthe group consisting of SEQ ID NO:178, SEQ ID NO:182, SEQ ID NO:186, SEQID NO:190, SEQ ID NO:194, SEQ ID NO:198, SEQ ID NO:202, SEQ ID NO:206,SEQ ID NO:210, SEQ ID NO:214, SEQ ID NO:218, SEQ ID NO:222, SEQ IDNO:226, SEQ ID NO:230, SEQ ID NO:234, SEQ ID NO:238, and SEQ ID NO: 242.In further embodiments, the present invention provides a Resistinantibody comprising (a) a heavy chain selected from the group consistingof SEQ ID NO:176, SEQ ID NO:180, SEQ ID NO:184, SEQ ID NO:188, SEQ IDNO:192, SEQ ID NO:196, SEQ ID NO:200, SEQ ID NO:204, SEQ ID NO:208, SEQID NO:212, SEQ ID NO:216, SEQ ID NO:220, SEQ ID NO:224, SEQ ID NO:228,SEQ ID NO:232, SEQ ID NO:236, and SEQ ID NO:240 and (b) a light chainselected from the group consisting of SEQ ID NO:178, SEQ ID NO:182, SEQID NO:186, SEQ ID NO:190, SEQ ID NO:194, SEQ ID NO:198, SEQ ID NO:202,SEQ ID NO:206, SEQ ID NO:210, SEQ ID NO:214, SEQ ID NO:218, SEQ IDNO:222, SEQ ID NO:226, SEQ ID NO:230, SEQ ID NO:234, SEQ ID NO:238, andSEQ ID NO: 242.

In several embodiments, the present invention provides Resistinantibodies that are also cross-reactive with Resistin Like Molecule Beta(RELMβ). In other embodiments, Resistin scfv are also cross-reactivewith RELMβ. In further embodiments, the antibodies and/or fragmentsthereof are recombinant.

In another aspect, the present invention provides methods for using theantibodies described herein. In particular embodiments, the presentinvention provides a method for treating a disease, disorder orcondition mediated by human Resistin in a patient comprising the step ofadministering to the patient an antibody an scfv described herein. Inspecific embodiments, the disease, disorder or condition is one or moreof pulmonary hypertension, cardiac hypertrophy and failure, asthma, lunginflammation, sepsis, acute lung injury, respiratory distress syndrome,pulmonary fibrosis, scleroderma, arteriosclerosis, chronic obstructivelung disease/emphysema, normal and abnormal wound healing, cancer, cellproliferation, stem cell growth and differentiation, diabeticretinopathy, and insulin resistance.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a graph showing the binding reactivity of Ab1 to humanResistin (hResistin) at absorbance 450 nm.

FIG. 2 is a graph showing the binding reactivity of Ab13 to humanResistin (hResistin) at absorbance 450 nm.

FIG. 3 is a graph showing the binding reactivity of Ab17 to humanResistin (hResistin) at absorbance 450 nm.

FIG. 4 is a graph showing the binding reactivity of Ab21 to humanResistin (hResistin) at absorbance 450 nm.

FIG. 5 is a graph showing the binding reactivity of Ab41 to humanResistin (hResistin) at absorbance 450 nm.

FIG. 6 is a graph showing the binding reactivity of R&D Ab to humanResistin (hResistin) at absorbance 450 nm.

FIG. 7 is a graph comparing the antibody reactivity of all theantibodies with 25 ng/ml hResistin. R&D Ab at 1 μg/ml; all other Abs 5μg/ml; values at 1 μg/ml R&D antibody used as value at 2 μg/ml is beyondabsorbance range for the assay.

FIG. 8 shows the plate layout for the hResistin antibody screen of Ab1,Ab13, Ab17, Ab21, Ab31 and R&D antibody. A-5, B-2, C-1, and D-0.5 μg/mlAb concentrations.

FIG. 9 shows the plate layout and resulting absorbance measurements forthe hResistin antibody screen of Ab1, Ab13, Ab17, Ab21, Ab31 and R&Dantibody.

FIG. 10 shows the constructs for human IgG1. CH123 is the heavy constantregion. CK is the light constant region.

FIG. 11 is a table showing a quality control rIgG ELISA of 17 anti-humanResistin IgGs.

FIG. 12 is a table showing ELISA results.

DETAILED DESCRIPTION OF THE INVENTION

It is understood that the present invention is not limited to theparticular methods and components, etc., described herein, as these mayvary. It is also to be understood that the terminology used herein isused for the purpose of describing particular embodiments only, and isnot intended to limit the scope of the present invention. It must benoted that as used herein and in the appended claims, the singular forms“a,” “an,” and “the” include the plural reference unless the contextclearly dictates otherwise. Thus, for example, a reference to a“protein” is a reference to one or more proteins, and includesequivalents thereof known to those skilled in the art and so forth.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Specific methods, devices, andmaterials are described, although any methods and materials similar orequivalent to those described herein can be used in the practice ortesting of the present invention.

All publications cited herein are hereby incorporated by referenceincluding all journal articles, books, manuals, published patentapplications, and issued patents. In addition, the meaning of certainterms and phrases employed in the specification, examples, and appendedclaims are provided. The definitions are not meant to be limiting innature and serve to provide a clearer understanding of certain aspectsof the present invention.

I. Definitions

The term “amino acid” refers to naturally occurring and synthetic aminoacids, as well as amino acid analogs and amino acid mimetics thatfunction in a manner similar to the naturally occurring amino acids.Naturally occurring amino acids are those encoded by the genetic code,as well as those amino acids that are later modified, for example,hydroxyproline, gamma-carboxyglutamate, and O-phosphoserine,phosphothreonine.

An “amino acid analog” refers to a compound that has the same basicchemical structure as a naturally occurring amino acid, i.e., a carbonthat is bound to a hydrogen, a carboxyl group, an amino group, and an Rgroup (e.g., homoserine, norleucine, methionine sulfoxide, methioninemethyl sulfonium), but that contains some alteration not found in anaturally occurring amino acid (e.g., a modified side chain). Aminoacids and analogs are well known in the art. Amino acids may be referredto herein by either their commonly known three letter symbols or by theone-letter symbols recommended by the IUPAC-IUB Biochemical NomenclatureCommission. Nucleotides, likewise, may be referred to by their commonlyaccepted single-letter codes. The term “amino acid mimetic” refers tochemical compounds that have a structure that is different from thegeneral chemical structure of an amino acid, but that function in amanner similar to a naturally occurring amino acid Amino acid analogsmay have modified R groups (for example, norleucine) or modified peptidebackbones, but retain the same basic chemical structure as a naturallyoccurring amino acid. In certain embodiments, an amino acid analog is aD-amino acid, a beta-amino acid, or an N-methyl amino acid.

By “antibody” is meant any immunoglobulin polypeptide, or fragmentthereof, having immunogen binding ability. As used herein, the terms“antibody fragments”, “fragment”, or “fragment thereof” refer to aportion of an intact antibody. Examples of antibody fragments include,but are not limited to, linear antibodies; single-chain antibodymolecules; Fc or Fc′ peptides, Fab and Fab fragments, and multispecificantibodies formed from antibody fragments. In most embodiments, theterms also refer to fragments that bind an antigen of a target molecule(e.g., Resistin) and can be referred to as “antigen-binding fragments.”

The term “conjugate” refers to a complex of two molecules linkedtogether, for example, linked together by a covalent bond. In oneembodiment, an antibody is linked to an effector molecule; for example,an antibody that specifically binds to Resistin covalently linked to aneffector molecule. The linkage can be by chemical or recombinant means.In one embodiment, the linkage is chemical, wherein a reaction betweenthe antibody moiety and the effector molecule has produced a covalentbond formed between the two molecules to form one molecule. A peptidelinker (short peptide sequence) can optionally be included between theantibody and the effector molecule. Because conjugates can be preparedfrom two molecules with separate functionalities, such as an antibodyand an effector molecule, they are also sometimes referred to as“chimeric molecules.”

The terms “conjugating,” “joining,” “bonding,” “labeling” or “linking”refer to making two molecules into one contiguous molecule; for example,linking two polypeptides into one contiguous polypeptide, or covalentlyattaching an effector molecule or detectable marker radionuclide orother molecule to a polypeptide, such as an scFv. In the specificcontext, the terms include reference to joining a ligand, such as anantibody moiety, to an effector molecule. The linkage can be either bychemical or recombinant means. “Chemical means” refers to a reactionbetween the antibody moiety and the effector molecule such that there isa covalent bond formed between the two molecules to form one molecule.

Conservative variants: “Conservative” amino acid substitutions are thosesubstitutions that do not substantially decrease the binding affinity ofan antibody for an antigen (for example, the binding affinity of anantibody for Resistin). For example, a human antibody that specificallybinds Resistin can include at most about 1, at most about 2, at mostabout 5, at most about 10, or at most about 1 5 conservativesubstitutions and specifically bind the Resistin polypeptide. The termconservative variation also includes the use of a substituted amino acidin place of an unsubstituted parent amino acid, provided that antibodyretains binding affinity for Resistin. Non-conservative substitutionsare those that reduce an activity or binding to Resistin.

Conservative amino acid substitution tables providing functionallysimilar amino acids are well known to one of ordinary skill in the art.The following six groups are examples of amino acids that are consideredto be conservative substitutions for one another:

1) Alanine (A), Serine (S), Threonine (T);

2) Aspartic acid (D), Glutamic acid (E);

3) Asparagine (N), Glutamine (Q);

4) Arginine (I), Lysine ( )

5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and

6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

An “effector molecule” means a molecule intended to have or produce adesired effect; for example, a desired effect on a cell to which theeffector molecule is targeted. Effector molecules include such moleculesas polypeptides, radioisotopes and small molecules. Non-limitingexamples of effector molecules include toxins, chemotherapeutic agentsand anti-angiogenic agents. The skilled artisan will understand thatsome effector molecules may have or produce more than one desiredeffect. In one example, an effector molecule is the portion of achimeric molecule, for example a chimeric molecule that includes adisclosed antibody or fragment thereof, that is intended to have adesired effect on a cell to which the chimeric molecule is targeted.

The term “epitope” or “antigenic determinant” are used interchangeablyherein and refer to that portion of an antigen capable of beingrecognized and specifically bound by a particular antibody. When theantigen is a polypeptide, epitopes can be formed both from contiguousamino acids and noncontiguous amino acids juxtaposed by tertiary foldingof a protein. Epitopes formed from contiguous amino acids are typicallyretained upon protein denaturing, whereas epitopes formed by tertiaryfolding are typically lost upon protein denaturing. An epitope typicallyincludes at least 3, and more usually, at least 5 or 8-10 amino acids ina unique spatial conformation. An antigenic determinant can compete withthe intact antigen (i.e., the “immunogen” used to elicit the immuneresponse) for binding to an antibody.

By “an effective amount” is meant the amount of a required to amelioratethe symptoms of a disease relative to an untreated patient. Theeffective amount of active compound(s) used to practice the presentinvention for therapeutic treatment of a vascular disease variesdepending upon the manner of administration, the age, body weight, andgeneral health of the subject. Ultimately, the attending physician orveterinarian will decide the appropriate amount and dosage regimen. Suchamount is referred to as an “effective” amount.

An “expression vector” is a nucleic acid construct, generatedrecombinantly or synthetically, bearing a series of specified nucleicacid elements that enable transcription of a particular gene in a hostcell. Typically, gene expression is placed under the control of certainregulatory elements, including constitutive or inducible promoters,tissue-preferred regulatory elements, and enhancers.

By “fragment” is meant a portion (e.g., at least about 5, 10, 25, 50,100, 125, 150, 200, 250, 300, 350, 400, or 500 amino acids or nucleicacids) of a protein or nucleic acid molecule that is substantiallyidentical to a reference protein or nucleic acid and retains at leastone biological activity of the reference. In some embodiments theportion retains at least 50%, 75%, or 80%, or more preferably 90%, 95%,or even 99% of the biological activity of the reference protein ornucleic acid described herein.

A “host cell” is any prokaryotic or eukaryotic cell that contains eithera cloning vector or an expression vector. This term also includes thoseprokaryotic or eukaryotic cells that have been genetically engineered tocontain the cloned gene(s) in the chromosome or genome of the host cell.

As used herein, “humanized” forms of non-human (e.g., murine) antibodiesare chimeric antibodies that contain minimal sequence, or no sequence,derived from non-human immunoglobulin. For the most part, humanizedantibodies are human immunoglobulins (recipient antibody) in whichresidues from a hypervariable region of the recipient are replaced byresidues from a hypervariable region of a non-human species (donorantibody) such as mouse, rat, rabbit or nonhuman primate having thedesired specificity, affinity, and capacity. In some instances, Fvframework region (FR) residues of the human immunoglobulin are replacedby corresponding non-human residues. Furthermore, humanized antibodiescan comprise residues that are not found in the recipient antibody or inthe donor antibody. These modifications are generally made to furtherrefine antibody performance. In general, the humanized antibody willcomprise substantially all of at least one, and typically two, variabledomains, in which all or substantially all of the hypervariable loopscorrespond to those of a nonhuman immunoglobulin and all orsubstantially all of the FR residues are those of a human immunoglobulinsequence. The humanized antibody can also comprise at least a portion ofan immunoglobulin constant region (Fc), typically that of a humanimmunoglobulin. Examples of methods used to generate humanizedantibodies are described in U.S. Pat. No. 5,225,539.

The term “human antibody” as used herein means an antibody produced by ahuman or an antibody having an amino acid sequence corresponding to anantibody produced by a human made using any of the techniques known inthe art. This definition of a human antibody includes intact orfull-length antibodies, fragments thereof, and/or antibodies comprisingat least one human heavy and/or light chain polypeptide such as, forexample, an antibody comprising murine light chain and human heavy chainpolypeptides.

“Hybrid antibodies” are immunoglobulin molecules in which pairs of heavyand light chains from antibodies with different antigenic determinantregions are assembled together so that two different epitopes or twodifferent antigens can be recognized and bound by the resultingtetramer.

The terms “isolated,” “purified,” or “biologically pure” refer tomaterial that is free to varying degrees from components which normallyaccompany it as found in its native state. Various levels of purity maybe applied as needed according to this invention in the differentmethodologies set forth herein; the customary purity standards known inthe art may be used if no standard is otherwise specified. Indeed, theterm “purified” does not require the material to be present in a formexhibiting absolute purity, exclusive of the presence of othercompounds. Thus, isolated nucleic acids, peptides and proteins includenucleic acids and proteins purified by standard purification methods.The term also embraces nucleic acids, peptides and proteins prepared byrecombinant expression in a host cell, as well as, chemicallysynthesized nucleic acids. A isolated nucleic acid, peptide or protein,for example an antibody, can be at least 50%, at least 60%, at least70%, at least 80%, at least 90%, at least 95%, at least 96%, at least97%, at least 98%, or at least 99% pure.

By “modulation” is meant a change (increase or decrease) in theexpression level or biological activity of a gene or polypeptide asdetected by standard methods known in the art. As used herein,modulation includes at least about 10% change, 25%, 40%, 50% or agreater change in expression levels or biological activity (e.g., about75%, 85%, 95% or more).

The term “mimetic” means an agent having a structure that is differentfrom the general chemical structure of a reference agent, but that hasat least one biological function of the reference.

The term “neutralizing antibody” refers to an antibody that is able tospecifically bind to a target protein in such a way as to inhibit abiological function associated with that target protein. In general, anyprotein that can perform this type of specific blocking activity isconsidered a neutralizing protein; neutralizing antibodies are thereforea specific class of neutralizing protein.

The term “nucleic acid” refers to an oligomer or polymer of ribonucleicacid or deoxyribonucleic acid, or analog thereof. This term includesoligomers consisting of naturally occurring bases, sugars, andintersugar (backbone) linkages as well as oligomers having non-naturallyoccurring portions which function similarly. Such modified orsubstituted oligonucleotides are often preferred over native formsbecause of properties such as, for example, enhanced stability in thepresence of nucleases.

Specific examples of some nucleic acids envisioned for this inventionmay contain phosphorothioates, phosphotriesters, methyl phosphonates,short chain alkyl or cycloalkyl intersugar linkages or short chainheteroatomic or heterocyclic intersugar linkages. Also preferred areoligonucleotides having morpholino backbone structures (Summerton, J. E.and Weller, D. D., U.S. Pat. No. 5,034,506). In other preferredembodiments, such as the protein-nucleic acid (PNA) backbone, thephosphodiester backbone of the oligonucleotide may be replaced with apolyamide backbone, the bases being bound directly or indirectly to theaza nitrogen atoms of the polyamide backbone (P. E. Nielsen et al.Science 199: 254, 1997). Other preferred oligonucleotides may containalkyl and halogen-substituted sugar moieties comprising one of thefollowing at the 2′ position: OH, SH, SCH₃, F, OCN, O(CH₂)_(n)NH₂ orO(CH₂)_(n)CH₃, where n is from 1 to about 10; C₁ to C₁₀ lower alkyl,substituted lower alkyl, alkaryl or aralkyl; Cl; Br; CN; CF₃; OCF₃; O—,S—, or N-alkyl; O—, S—, or N-alkenyl; SOCH₃; SO₂CH₃; ONO₂; NO₂; N₃; NH₂;heterocycloalkyl; heterocycloalkaryl; aminoalkylamino; polyalkylamino;substituted silyl; an RNA cleaving group; a conjugate; a reporter group;an intercalator; a group for improving the pharmacokinetic properties ofan oligonucleotide; or a group for improving the pharmacodynamicproperties of an oligonucleotide and other substituents having similarproperties. Oligonucleotides may also have sugar mimetics such ascyclobutyls in place of the pentofuranosyl group. Other preferredembodiments may include at least one modified base form. Some specificexamples of such modified bases include 2-(amino)adenine,2-(methylamino)adenine, 2-(imidazolylalkyl)adenine,2-(aminoalklyamino)adenine, or other heterosubstituted alkyladenines.

The term “operably linked” means that a first polynucleotide ispositioned adjacent to a second polynucleotide that directstranscription of the first polynucleotide when appropriate molecules(e.g., transcriptional activator proteins) are bound to the secondpolynucleotide.

By “recombinant” is meant the product of genetic engineering or chemicalsynthesis. By “positioned for expression” is meant that thepolynucleotide of the present invention (e.g., a DNA molecule) ispositioned adjacent to a DNA sequence that directs transcription andtranslation of the sequence (i.e., facilitates the production of, forexample, a recombinant protein of the present invention, or an RNAmolecule).

The term “reference” means a standard or control condition.

The terms “specifically binds to,” “specific for,” and relatedgrammatical variants refer to that binding which occurs between suchpaired species as antibody/antigen, aptamer/target, enzyme/substrate,receptor/agonist and lectin/carbohydrate which may be mediated bycovalent or non-covalent interactions or a combination of covalent andnon-covalent interactions. When the interaction of the two speciesproduces a non-covalently bound complex, the binding which occurs istypically electrostatic, hydrogen-bonding, or the result of lipophilicinteractions. Accordingly, in certain embodiments, “specific binding”occurs between a paired species where there is interaction between thetwo which produces a bound complex having the characteristics of, forexample, an antibody/antigen. In particular, the specific binding ischaracterized by the binding of one member of a pair to a particularspecies and to no other species within the family of compounds to whichthe corresponding member of the binding member belongs. Thus, forexample, an antibody typically binds to a single epitope and to no otherepitope within the family of proteins. In some embodiments, specificbinding between an antigen and an antibody will have a binding affinityof at least 10⁻⁶ M. In other embodiments, the antigen and antibody willbind with affinities of at least 10⁻⁷ M, 10⁻⁸ M to 10⁻⁹ M, 10⁻¹⁰ M,10⁻¹¹ M, or 10⁻¹² M. In certain embodiments, the term refers to amolecule (e.g., an antibody) that binds to a target (e.g., Resistin)with at least five-fold greater affinity as compared to any non-targets,e.g., at least 10-, 20-, 50-, or 100-fold greater affinity.

By “subject” is meant a mammal, including, but not limited to, a humanor non-human mammal, such as a bovine, equine, canine, ovine, or feline.

By “substantially identical” is meant a protein or nucleic acid moleculeexhibiting at least 50% identity to a reference amino acid sequence (forexample, any one of the amino acid sequences described herein) ornucleic acid sequence (for example, any one of the nucleic acidsequences described herein). Preferably, such a sequence is at least60%, more preferably 80% or 85%, and most preferably 90%, 95% or even99% identical at the amino acid level or nucleic acid to the sequenceused for comparison.

Sequence identity is typically measured using sequence analysis software(for example, Sequence Analysis Software Package of the GeneticsComputer Group, University of Wisconsin Biotechnology Center, 1710University Avenue, Madison, Wis. 53705, BLAST, BESTFIT, GAP, orPILEUP/PRETTYBOX programs). Such software matches identical or similarsequences by assigning degrees of homology to various substitutions,deletions, and/or other modifications. Conservative substitutionstypically include substitutions within the following groups: glycine,alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid,asparagine, glutamine; serine, threonine; lysine, arginine; andphenylalanine, tyrosine. In an exemplary approach to determining thedegree of identity, a BLAST program may be used, with a probabilityscore between e.sup.-3 and e.sup.-100 indicating a closely relatedsequence.

By “transformed cell” is meant a cell into which (or into an ancestor ofwhich) has been introduced, by means of recombinant DNA techniques, apolynucleotide molecule encoding (as used herein) a protein of thepresent invention.

II. Antibodies to Resistin and Resistin-Like Molecule Beta (RELMB)

The present invention provides antibodies to Resistin. In someembodiments, the antibodies are also cross-reactive with Resistin-LikeMolecule Beta (RELMB). An “antibody” is a polypeptide ligand includingat least the complementarity determining regions (CDRs) of a light chainor heavy chain immunoglobulin variable region which specifically bindsan epitope of an antigen or a fragment thereof. Antibodies includeintact immunoglobulins and the variants of them well known in the art,such as Fab′, F(ab)′2 fragments, single chain Fv proteins (scFv), anddisulfide stabilized Fv proteins (dsFv). A scFvprotein is a fusionprotein in which a light chain variable region of an antibody and aheavy chain variable region of an antibody are bound by a linker, whilein dsFvs, the chains have been mutated to introduce a disulfide bond tostabilize the association of the chains. The term “antibody” alsoincludes genetically engineered forms such as chimeric antibodies (forexample, humanized murine antibodies) and heteroconjugateantibodies(such as, bispecific antibodies).

Typically, a naturally occurring immunoglobulin has heavy (H) chains andlight (L) chains interconnected by disulfide bonds. There are two typesof light chains, lambda (λ) and kappa (κ). There are five main heavychain classes (or isotypes) which determine the functional activity ofan antibody molecule: IgM, IgD, IgG, IgA and IgE.

Each heavy and light chain contains a constant region and a variableregion (the regions are also known as domains). References to “VH” or“VH” refer to the variable region of an immunoglobulin heavy chain,including that of an Fv, scFv, dsFv or Fab. References to “VL” or “VL”refer to the variable region of an immunoglobulin light chain, includingthat of an Fv, scFv, dsFv or Fab. In combination, the heavy and thelight chain variable regions specifically bind the antigen. Light andheavy chain variable regions contain a framework region interrupted bythree hypervariable regions, also called complementarity-determiningregions or CDRs. The extent of the framework region and CDRs have beendefined (see, for example, Kabat et al., (1991) Sequences of Proteins ofImmunological Interest, 5lh Edition, U.S. Department of Health and HumanServices, Public Health Service, National Institutes of Health,Bethesda, Md. (NIH Publication No. 91-3242), which is herebyincorporated by reference). The sequences of the framework regions ofdifferent light or heavy chains are relatively conserved within aspecies. The framework region of an antibody, that is the combinedframework regions of the constituent light and heavy chains, serves toposition and align the CDRs in three-dimensional space.

The CDRs are primarily responsible for binding to an epitope of anantigen. The precise amino acid sequence boundaries of a given CDR canbe readily determined using any of a number of well-known schemes,including those described by Kabat et al. (1991), “Sequences of Proteinsof Immunological Interest,” 5th Ed. Public Health Service, NationalInstitutes of Health, Bethesda, Md. (“Kabat” numbering scheme), andAl-Lazikani et al., (1997) JMB 273,927-948 (“Chothia” numbering scheme).The CDRs of each chain are typically referred to as CDR1, CDR2, andCDR3, numbered sequentially starting from the N-terminus, and are alsotypically identified by the chain in which the particular CDR islocated. Thus, a HCDR1 is the CDR 1 from the variable domain of theheavy chain of the antibody in which it is found, whereas a LCDR 1 isthe CDR1 from the variable domain of the light chain of the antibody inwhich it is found. An antibody that specifically binds an antigen ofinterest has a specific VH region and VL region sequence, and thusspecific CDR sequences. Antibodies with different specificities (due todifferent combining sites for different antigens) have different CDRs.Although it is the CDRs that vary from antibody to antibody, only alimited number of amino acid positions within the CDRs are directlyinvolved in antigen binding. These positions within the CDRs are calledspecificity determining residues (SDRs).

A single-chain antibody (scFv) is a genetically engineered moleculecontaining the VH and VL domains of one or more antibody(ies) linked bya suitable polypeptide linker as a genetically fused single chainmolecule. Diabodies are bivalent, bispecific antibodies in which VH andVL domains are expressed on a single polypeptide chain, but using alinker that is too short to allow for pairing between the two domains onthe same chain, thereby forcing the domains to pair with complementarydomains of another chain and creating two antigen binding sites. Achimeric antibody is an antibody that contains one or more regions fromone antibody and one or more regions from one or more other antibodies.An antibody may have one or more binding sites. If there is more thanone binding site, the binding sites may be identical to one another ormay be different. For instance, a naturally-occurring immunoglobulin hastwo identical binding sites, a single-chain antibody or Fab fragment hasone binding site, while a bispecific or bifunctional antibody has twodifferent binding sites.

The antibodies disclosed herein specifically bind only to a definedtarget (or multiple targets, in the case of a bi-specific antibody).Thus, an antibody that specifically binds to Resistin is an antibodythat binds substantially to Resistin, including cells or tissueexpressing Resistin, substrate to which the Resistin is attached, orResistin in a biological specimen. It is, of course, recognized that acertain degree of non-specific interaction may occur between an antibodyor conjugate including an antibody (such as an antibody thatspecifically binds Resistin or conjugate including such antibody) and anon-target (such as a cell that does not express Resistin). Typically,specific binding results in a much stronger association between theantibody and protein or cells bearing the antigen than between theantibody and protein or cells lacking the antigen. Specific bindingtypically results in greater than 2-fold, such as greater than 5-fold,greater than 10-fold, or greater than 100-fold increase in amount ofbound antibody (per unit time) to a protein including the epitope orcell or tissue expressing the target epitope as compared to a protein orcell or tissue lacking this epitope.

In one embodiment, an antibody that binds Resistin is monoclonal.Alternatively, the Resistin antibody is a polyclonal antibody. Thepreparation and use of polyclonal antibodies are also known the skilledartisan. The present invention also encompasses hybrid antibodies, inwhich one pair of heavy and light chains is obtained from a firstantibody, while the other pair of heavy and light chains is obtainedfrom a different second antibody. Such hybrids may also be formed usinghumanized heavy and light chains. Such antibodies are often referred toas “chimeric” antibodies.

In general, intact antibodies are said to contain “Fc” and “Fab”regions. The Fc regions are involved in complement activation and arenot involved in antigen binding. An antibody from which the Fc′ regionhas been enzymatically cleaved, or which has been produced without theFc′ region, designated an “F(abα)₂” fragment, retains both of theantigen binding sites of the intact antibody. Similarly, an antibodyfrom which the Fc region has been enzymatically cleaved, or which hasbeen produced without the Fc region, designated an “Fab′” fragment,retains one of the antigen binding sites of the intact antibody. Fabαfragments consist of a covalently bound antibody light chain and aportion of the antibody heavy chain, denoted “Fd.” The Fd fragments arethe major determinants of antibody specificity (a single Fd fragment maybe associated with up to ten different light chains without alteringantibody specificity). Isolated Fd fragments retain the ability tospecifically bind to immunogenic epitopes.

Monoclonal antibodies can be prepared using hybridoma methods, such asthose described by Kohler and Milstein (1975) Nature 256:495. Using thehybridoma method, a mouse, hamster, or other appropriate host animal, isimmunized as described above to elicit the production by lymphocytes ofantibodies that will specifically bind to an immunizing antigen.Alternatively, lymphocytes can be immunized in vitro. Followingimmunization, the lymphocytes are isolated and fused with a suitablemyeloma cell line using, for example, polyethylene glycol, to formhybridoma cells that can then be selected away from unfused lymphocytesand myeloma cells. Hybridomas that produce monoclonal antibodiesdirected specifically against a chosen antigen as determined byimmunoprecipitation, immunoblotting, or by an in vitro binding assaysuch as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay(ELISA) can then be propagated either in vitro culture using standardmethods (Goding, Monoclonal Antibodies: Principles and Practice,Academic Press, 1986) or in vivo as ascites tumors in an animal Themonoclonal antibodies can then be purified from the culture medium orascites fluid as described for polyclonal antibodies above.

Alternatively monoclonal antibodies can also be made using recombinantDNA methods as described in U.S. Pat. No. 4,816,567. The polynucleotidesencoding a monoclonal antibody are isolated, such as from mature B-cellsor hybridoma cell, such as by RT-PCR using oligonucleotide primers thatspecifically amplify the genes encoding the heavy and light chains ofthe antibody, and their sequence is determined using conventionalprocedures. The isolated polynucleotides encoding the heavy and lightchains are then cloned into suitable expression vectors, which whentransfected into host cells such as E. coli cells, simian COS cells,Chinese hamster ovary (CHO) cells, or myeloma cells that do nototherwise produce immunoglobulin protein, monoclonal antibodies aregenerated by the host cells. Also, recombinant monoclonal antibodies orfragments thereof of the desired species can be isolated from phagedisplay libraries as described (McCafferty et al., 1990, Nature,348:552-554; Clackson et al., 1991, Nature, 352:624-628; and Marks etal., 1991, J. Mol. Biol., 222:581-597).

The polynucleotide(s) encoding a monoclonal antibody can further bemodified in a number of different ways using recombinant DNA technologyto generate alternative antibodies. In one embodiment, the constantdomains of the light and heavy chains of, for example, a mousemonoclonal antibody can be substituted 1) for those regions of, forexample, a human antibody to generate a chimeric antibody or 2) for anon-immunoglobulin polypeptide to generate a fusion antibody. In otherembodiments, the constant regions are truncated or removed to generatethe desired antibody fragment of a monoclonal antibody. Furthermore,site-directed or high-density mutagenesis of the variable region can beused to optimize specificity, affinity, etc. of a monoclonal antibody.

In some embodiments, of the present invention the monoclonal antibodyagainst Resistin is a humanized antibody. Humanized antibodies areantibodies that contain minimal sequences from non-human (e.g., murine)antibodies within the variable regions. In practice, humanizedantibodies are typically human antibodies with minimum to no non-humansequences. A human antibody is an antibody produced by a human or anantibody having an amino acid sequence corresponding to an antibodyproduced by a human.

Humanized antibodies can be produced using various techniques known inthe art. An antibody can be humanized by substituting the CDR of a humanantibody with that of a non-human antibody (e.g., mouse, rat, rabbit,hamster, etc.) having the desired specificity, affinity, and capability(Jones et al., 1986, Nature, 321:522-525; Riechmann et al., 1988,Nature, 332:323-327; Verhoeyen et al., 1988, Science, 239:1534-1536).The humanized antibody can be further modified by the substitution ofadditional residue either in the Fv framework region and/or within thereplaced non-human residues to refine and optimize antibody specificity,affinity, and/or capability.

Human antibodies can be directly prepared using various techniques knownin the art. Immortalized human B lymphocytes immunized in vitro orisolated from an immunized individual that produce an antibody directedagainst a target antigen can be generated (See, for example, Cole etal., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77(1985); Boerner et al., 1991, J. Immunol., 147 (1):86-95; and U.S. Pat.No. 5,750,373). Also, the human antibody can be selected from a phagelibrary, where that phage library expresses human antibodies (Vaughan etal., 1996, Nature Biotechnology, 14:309-314; Sheets et al., 1998, PNAS,95:6157-6162; Hoogenboom and Winter, 1991, J. Mol. Biol., 227:381; Markset al., 1991, J. Mol. Biol., 222:581). Humanized antibodies can also bemade in transgenic mice containing human immunoglobulin loci that arecapable upon immunization of producing the full repertoire of humanantibodies in the absence of endogenous immunoglobulin production. Thisapproach is described in U.S. Pat. Nos. 5,545,807; 5,545,806; 5,569,825;5,625,126; 5,633,425; and 5,661,016.

In certain embodiments of the invention, it may be desirable to use anantibody fragment, rather than an intact antibody. Various techniquesare known for the production of antibody fragments. Traditionally, thesefragments are derived via proteolytic digestion of intact antibodies(for example Morimoto et al., 1993, Journal of Biochemical andBiophysical Methods 24:107-117 and Brennan et al., 1985, Science,229:81). However, these fragments are now typically produced directly byrecombinant host cells as described above. Thus Fab, Fv, and scFvantibody fragments can all be expressed in and secreted from E. coli orother host cells, thus allowing the production of large amounts of thesefragments. Alternatively, such antibody fragments can be isolated fromthe antibody phage libraries discussed above. The antibody fragment canalso be linear antibodies as described in U.S. Pat. No. 5,641,870, forexample, and can be monospecific or bispecific. Other techniques for theproduction of antibody fragments will be apparent.

The present invention further embraces variants and equivalents whichare substantially homologous to the chimeric, humanized and humanantibodies, or antibody fragments thereof, set forth herein. These cancontain, for example, conservative substitution mutations, i.e., thesubstitution of one or more amino acids by similar amino acids. Forexample, conservative substitution refers to the substitution of anamino acid with another within the same general class such as, forexample, one acidic amino acid with another acidic amino acid, one basicamino acid with another basic amino acid or one neutral amino acid byanother neutral amino acid. What is intended by a conservative aminoacid substitution is well known in the art.

Without further elaboration, it is believed that one skilled in the art,using the preceding description, can utilize the present invention tothe fullest extent. The following examples are illustrative only, andnot limiting of the remainder of the disclosure in any way whatsoever.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how thecompounds, compositions, articles, devices, and/or methods described andclaimed herein are made and evaluated, and are intended to be purelyillustrative and are not intended to limit the scope of what theinventors regard as their invention. Efforts have been made to ensureaccuracy with respect to numbers (e.g., amounts, temperature, etc.) butsome errors and deviations should be accounted for herein. Unlessindicated otherwise, parts are parts by weight, temperature is indegrees Celsius or is at ambient temperature, and pressure is at or nearatmospheric. There are numerous variations and combinations of reactionconditions, e.g., component concentrations, desired solvents, solventmixtures, temperatures, pressures and other reaction ranges andconditions that can be used to optimize the product purity and yieldobtained from the described process. Only reasonable and routineexperimentation will be required to optimize such process conditions.

Example 1 Human Resistin Antibody Screen (ELISA)

Purpose. Screen five candidate antibodies to human resistin for prospectat use in neutralization experiments against human resistin.

Background. A direct-coat ELISA was performed (resistin coated to wellsof plate, then run antibodies against immobilized antigen, see Protocol1), identifying 12 antibodies to have “positive” reactivity, albeit tovarying degree. From these 12, five have been selected for furtheranalysis (all 17 candidate antibodies showed some level of success inbinding human resistin, via immunoprecipitation); these five are thoseshowing highest positive reactivity in the matrix: Ab1, Ab13, Ab17,Ab21, and Ab41. For potential as neutralizing antibodies, recognition ofimmobilized antigen is not sufficient. Direct evaluation of theantibodies with neutralization experiments are costly and can beinefficient as controls become detailed; thus, sandwich-type ELISAbecomes a valid tool for evaluating which antibodies may be best suitedfor further analysis. Furthermore, this ELISA assay should provideuseful information with regard to effective concentration(s) of thetested antibodies against a standard human resistin protein.

General Description of ELISA. Indirect ELISA utilizes a set of twoantibodies that recognize the target antigen arranged such that oneantibody (capture antibody) is pre-adsorbed to the wells of a 96-wellplate. A solution containing the antigen of interest is passed over theadsorbed capture antibody and some amount of antigen is usually removedfrom solution. The second antibody recognizing the antigen of interestis then added to the wells, and again some level of previously capturedantigen is recognized by this second (detection antibody) antibody.Finally, a secondary antibody (commonly HRP tagged) that recognized thehost species for production of the detection antibody is added to thewells, and substrate added for eventual quantification. There areseveral variations to this method.

Points of Note:

*It is imperative that the capture and detection antibodies are a“matched pair” in that they do not compete with one another for bindingthe target antigen, as this would deliver a false negative reading.Indirect/sandwich type ELISA kits from commercial sources have beenvetted for this condition and will be labeled as such. Additionally, ifthe capture and detection antibody are from monoclonal and polyclonalsources, it is most common to use the monoclonal for capture, and thepolyclonal as the detection antibody for maximum specificity.

*In the case of utilization of the indirect/sandwich ELISA forevaluation of a capture/detection antibody, it is possible to use anon-antigen specific antibody paired with the antibody under evaluation.For example, if the antigen can be had in a FLAG or HIS tag, orsomething similar, and provided the antibody under evaluation binding isnot interrupted by the presence of such a tag or addition, then thesecond antibody (commonly the detection antibody) can be directed towardthis additional motif (FLAG, HIS). This allows evaluation of antibodieswithout having to have more than one antibody directed toward the targetantigen. Caution must be exercised here with regard to adequate controlsfor non-specific binding (wells with no capture antibody, wells with noantigen solution, wells with no detection antibody, etc.).

Specific Background on this ELISA:

Using part of R&D Systems Six-Pack (Cat #SRSN00; R&D Protocol):

-   -   Wash Buffer Concentrate #895003 (20 mL diluted to 500 mL with DI        H2O)    -   hResistin standard #892671 (reconstituted with 1.0 mL DI H2O as        per R&D Protocol)    -   hResistin conjugate #892670 (used as detection antibody as        packaged)    -   Substrate solution #895000 and 895001 (11 mL each Solution A and        B, mixed and used within 15 minutes as per R&D Protocol)    -   Stop Solution #895032 (used as packaged as per R&D Protocol)

For comparison, using a commercial monoclonal Ms anti-huRes (R&D #13591clone 184305) as one of coated capture antibodies

96-well plate from R&D: #DY990, Lot 301409

Using range of resistin concentrations (normal is 25-50 ng/mL) to showeffectiveness in detecting within, above, and below normal range.

Protocol:

Day 1:

1. Prepare ELISA coating buffer:

-   -   0.05 M Na2CO3/NaHCO3 (in one embodiment, 0.05 M is used;        0.05-0.1 M is common range)    -   2.173 g Na2CO3    -   3.528 g NaHCO3    -   Dissolve both in 1 L beaker, ˜700 mL DI H2O. Correct pH to ˜9.5        (9.51)    -   Q.S. to 1 L, re-check and adjust pH (9.54, 9.51 final)    -   Transfer to 1 L bottle, label, store at 4 C (Cold Room)

2. Prepare dilutions of 5 candidate capture antibodies (Ab1, Ab13, Ab17,Ab21, Ab41) for final concentrations: 5, 2, 1, 0.5 g/mL (Range is 1-12g/mL in commercial kits, but for testing purposes and as some antibodyquantities are limited, used 0.5-5 g/mL; 0.5-1.0 mL each prepared: 4wells per concentration, 100 L/wl). Also made R&D Ab at 2, 1, 0.5 g/mL(leave 4 wells for ‘blank,’ see Plate Layout).

3. Add 100 L/well as per Plate Layout, each of 4 candidate antibodies at4 dilutions in 4 wells each; Comparison R&D antibody at 3 dilutions in 4wells each. Column 12, rows A-D left blank, loaded only coating buffer.*Used eppendorf repeating pipette for delivery of solutions.

4. Covered with sealing adhesive strip, put on rotator in cold room at4° C. overnight.

Day 2:

5. Remove coating antibody and solution by inverting in sink, then wash4 times with wash buffer, removing all liquid each time. After finalrinse, invert on paper towels to blot excess liquid before addingBlocking Solution.

6. Add 250 L Blocking Solution: 2% BSA (w/v) in PBS-T (PBS [Commercial,Gibco] with 0.05% Tween 20). Cover with new sealing adhesive strip,block overnight at 4 C in cold room on rotator again.

*In some embodiments, probably not necessary to block overnight, was dueto concurrent experiments in this case; 2 h at RT to overnight at 4 C isacceptable.

Day 3:

7. Set up dilutions of hResistin standard protein (as directed in R&DProtocol *Consult Protocol from R&D): 100, 25, 10, 1 ng/mL in PBST (Seestep 6 for preparation of PBST). Add 100 L each to appropriate wells(see plate layout). Add new sealing adhesive strip, incubate at roomtemperature 2 hours on rotator.

8. Remove hResistin protein and wash 4-5× as before. Add hRes conjugateantibody, 200 L/well ALL WELLS. Add new sealing adhesive strip, incubateat room temperature 2 hours on rotator. *With 5 minutes remaining,combine equal volumes of Substrate solution A and B, mix, let sit (mustbe used within 15 minutes of mixing) and protect from light.

9. Remove hResistin conjugate and wash 4-5× as above. Add 200 L/well ALLWELLS mixed Substrate Solution, cover with new adhesive sealing strip,tap sides gently to mix. Cover with foil to protect from light and placein drawer for 30 minutes.

10. Remove adhesive strip, add 50 L Stop Solution per well ALL WELLS,tap gently to mix. Read within 30 minutes at 450 and either 540 or 570nm (subtract reading at 540 or 570 from 450 for correction).

Results

Antibodies 13, 17, 21, and 41 all show some reactivity in bindinghResistin. Ab1 did not show sufficient reactivity to be considered abovebaseline. Overall, all five tested antibodies were far less competent inbinding and pulling hResistin out of solution in comparison to thecommercial R&D antibody.

FIGS. 1-6 show corrected absorbance at 450 nm of each of the five testand one comparison antibody, note that all test antibodies are shownwith the same Z/Absorbance axis, but R&D antibody (FIG. 6) is muchlarger axis. FIG. 7 compares all six antibodies present on the plate, atmaximum coating antibody concentration and at 25 ng/mL hResistin added(corresponds to maxima of most test antibodies; see individual figures).

Discussion

Antibodies 13, 17, 21, and 41 all show saturation in huRes binding at˜25 ng/mL, with antibodies concentration at 5 g/mL.

Example 2: Convert scfv into Fully Human IgG1. The seventeen scfv(nucleotide sequences found in SEQ ID NO:1, SEQ ID NO:11, SEQ ID NO:21,SEQ ID NO:31, SEQ ID NO:41, SEQ ID NO:51, SEQ ID NO:61, SEQ ID NO:71,SEQ ID NO:81, SEQ ID NO:91, SEQ ID NO:101, SEQ ID NO:111, SEQ ID NO:121,SEQ ID NO:131, SEQ ID NO:141, SEQ ID NO:151 and SEQ ID NO:161; aminoacid sequences found in SEQ ID NO:2, SEQ ID NO:12, SEQ ID NO:22, SEQ IDNO:32, SEQ ID NO:42, SEQ ID NO:52, SEQ ID NO:62, SEQ ID NO:72, SEQ IDNO:82, SEQ ID NO:92, SEQ ID NO:102, SEQ ID NO:112, SEQ ID NO:122, SEQ IDNO:132, SEQ ID NO:142, SEQ ID NO:152, SEQ ID NO:162) converted intofully human IgG1. Briefly, the cDNA of the variable heavy (SEQ ID NO:3,SEQ ID NO:13, SEQ ID NO:23, SEQ ID NO:33, SEQ ID NO:43, SEQ ID NO:53,SEQ ID NO:63, SEQ ID NO:73, SEQ ID NO:83, SEQ ID NO:93, SEQ ID NO:103,SEQ ID NO:113, SEQ ID NO:123, SEQ ID NO:133, SEQ ID NO:143, SEQ IDNO:153, SEQ ID NO:163) and the variable light (SEQ ID NO:7, SEQ IDNO:17, SEQ ID NO:27, SEQ ID NO:37, SEQ ID NO:47, SEQ ID NO:57, SEQ IDNO:67, SEQ ID NO:77, SEQ ID NO:87, SEQ ID NO:97, SEQ ID NO:107, SEQ IDNO:117, SEQ ID NO:127, SEQ ID NO:137, SEQ ID NO:147, SEQ ID NO:157, SEQID NO:167) were subcloned into an IgG-expressing vector. Two separatevectors for the light chain and heavy chain were used. The vectors weretransiently transfected in 293 cells, and the expressed IgGs werepurified with Protein A or G. The sequences for the heavy constantregion are shown in SEQ ID NO:171 (nucleotide) and SEQ ID NO:172 (aminoacid). The sequences for the light constant region are shown in SEQ IDNO:173 (nucleotide) and SEQ ID NO:174 (amino acid). See also FIG. 10.

Example 3: rIgGs Show Cross-Reactivity to RELMβ. As shown in FIG. 11,the supernatant of cells expressing 17 rIgG was subjected to QC ELISA inwhich two targets were coated in parallel. As a result, 4 IgGs (Clones1, 2, 13 and 18) bind to two targets positively. hRELMβ3: 0.5 μg/well;hResistin: 5 μg/well; secondary antibody: FITC-goat anti-human IgG pAb.

1. An isolated, recombinant antibody that binds human Resistin.
 2. Anucleotide sequence that encodes an antibody that binds human Resistin.3. An amino acid sequence that encodes an antibody that binds humanResistin.
 4. The antibody of claim 1, wherein the antibody is a singlechain variable fragment (scFv), a dimeric scFv, a Fab, a Fab′, a F(ab′)2fragment or a full length antibody.
 5. The antibody of claim 1, whereinthe antibody comprises a variable heavy chain comprising SEQ ID NO:3,SEQ ID NO:13, SEQ ID NO:23, SEQ ID NO:33, SEQ ID NO:43, SEQ ID NO:53,SEQ ID NO:63, SEQ ID NO:73, SEQ ID NO:83, SEQ ID NO:93, SEQ ID NO:103,SEQ ID NO:113, SEQ ID NO:123, SEQ ID NO:133, SEQ ID NO:143, SEQ IDNO:153, SEQ ID NO:163, or fragments thereof.
 6. The antibody of claim 1,wherein the antibody comprises a variable heavy chain that is at least90% identical to SEQ ID NO:3, SEQ ID NO:13, SEQ ID NO:23, SEQ ID NO:33,SEQ ID NO:43, SEQ ID NO:53, SEQ ID NO:63, SEQ ID NO:73, SEQ ID NO:83,SEQ ID NO:93, SEQ ID NO:103, SEQ ID NO:113, SEQ ID NO:123, SEQ IDNO:133, SEQ ID NO:143, SEQ ID NO:153, SEQ ID NO:163, or fragmentsthereof.
 7. The antibody of claim 1, wherein the antibody comprises alight chain comprising SEQ ID NO:7, SEQ ID NO:17, SEQ ID NO:27, SEQ IDNO:37, SEQ ID NO:47, SEQ ID NO:57, SEQ ID NO:67, SEQ ID NO:77, SEQ IDNO:87, SEQ ID NO:97, SEQ ID NO:107, SEQ ID NO:117, SEQ ID NO:127, SEQ IDNO:137, SEQ ID NO:147, SEQ ID NO:157, SEQ ID NO:167, or fragmentsthereof.
 8. The antibody of claim 1, wherein the antibody comprises alight chain that is at least 90% identical to SEQ ID NO:7, SEQ ID NO:17,SEQ ID NO:27, SEQ ID NO:37, SEQ ID NO:47, SEQ ID NO:57, SEQ ID NO:67,SEQ ID NO:77, SEQ ID NO:87, SEQ ID NO:97, SEQ ID NO:107, SEQ ID NO:117,SEQ ID NO:127, SEQ ID NO:137, SEQ ID NO:147, SEQ ID NO:157, SEQ IDNO:167, or fragments thereof.
 9. The antibody of claim 1, wherein thevariable domain of the heavy chain comprises one or more complementaritydetermining regions (CDRs) selected from the group consisting of SEQ IDNO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:14, SEQ ID NO:15, SEQ IDNO:16, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:34, SEQ IDNO:35, SEQ ID NO:36, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ IDNO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:64, SEQ ID NO:65, SEQ IDNO:66, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:4, SEQ IDNO:85, SEQ ID NO:86, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ IDNO:104, SEQ ID NO:105, SEQ ID NO:106, SEQ ID NO:114, SEQ ID NO:115, SEQID NO:116, SEQ ID NO:124, SEQ ID NO:125, SEQ ID NO:126, SEQ ID NO:134,SEQ ID NO:135, SEQ ID NO:136, SEQ ID NO:144, SEQ ID NO:145, SEQ IDNO:146, SEQ ID NO:154, SEQ ID NO:155, SEQ ID NO:156, SEQ ID NO:164, SEQID NO:165, SEQ ID NO:166, and fragments thereof.
 10. The antibody ofclaim 1, wherein the variable domain of the heavy chain comprises one ormore complementarity determining regions (CDRs) that are at least 90%identical to a CDR selected from the group consisting of SEQ ID NO:4,SEQ ID NO:15, SEQ ID NO:6, SEQ ID NO:14, SEQ ID NO:5, SEQ ID NO:16, SEQID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:34, SEQ ID NO:35, SEQ IDNO:36, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:54, SEQ IDNO:55, SEQ ID NO:56, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ IDNO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:4, SEQ ID NO:85, SEQ IDNO:86, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:104, SEQ IDNO:105, SEQ ID NO:106, SEQ ID NO:114, SEQ ID NO:115, SEQ ID NO:116, SEQID NO:124, SEQ ID NO:125, SEQ ID NO:126, SEQ ID NO:134, SEQ ID NO:135,SEQ ID NO:136, SEQ ID NO:144, SEQ ID NO:145, SEQ ID NO:146, SEQ IDNO:154, SEQ ID NO:155, SEQ ID NO:156, SEQ ID NO:164, SEQ ID NO:165, SEQID NO:166, and fragments thereof.
 11. The antibody of claim 1, whereinthe variable domain of the light chain comprises one or more CDRsselected from the group consisting of SEQ ID NO:8, SEQ ID NO:9, SEQ IDNO:10, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:28, SEQ IDNO:29, SEQ ID NO:30, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ IDNO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:58, SEQ ID NO:59, SEQ IDNO:60, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:78, SEQ IDNO:79, SEQ ID NO:80, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ IDNO:98, SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:108, SEQ ID NO:109, SEQ IDNO:110, SEQ ID NO:118, SEQ ID NO:119, SEQ ID NO:120, SEQ ID NO:128, SEQID NO:129, SEQ ID NO:130, SEQ ID NO:138, SEQ ID NO:139, SEQ ID NO:140,SEQ ID NO:148, SEQ ID NO:149, SEQ ID NO:150, SEQ ID NO:158, SEQ IDNO:159, SEQ ID NO:160, SEQ ID NO:168, SEQ ID NO:169, SEQ ID NO:170, andfragments thereof.
 12. The antibody of claim 1, wherein the variabledomain of the light chain comprises one or more CDRs that are at least90% identical to a CDR selected from the group consisting of SEQ IDNO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:18, SEQ ID NO:19, SEQ IDNO:20, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:38, SEQ IDNO:39, SEQ ID NO:40, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ IDNO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:68, SEQ ID NO:69, SEQ IDNO:70, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:88, SEQ IDNO:89, SEQ ID NO:90, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:100, SEQ IDNO:108, SEQ ID NO:109, SEQ ID NO:110, SEQ ID NO:118, SEQ ID NO:119, SEQID NO:120, SEQ ID NO:128, SEQ ID NO:129, SEQ ID NO:130, SEQ ID NO:138,SEQ ID NO:139, SEQ ID NO:140, SEQ ID NO:148, SEQ ID NO:149, SEQ IDNO:150, SEQ ID NO:158, SEQ ID NO:159, SEQ ID NO:160, SEQ ID NO:168, SEQID NO:169, SEQ ID NO:170, and fragments thereof.
 13. An scfv that bindshuman Resistin, wherein the scfv is encoded by SEQ ID NO:1, SEQ IDNO:11, SEQ ID NO:21, SEQ ID NO:31, SEQ ID NO:41, SEQ ID NO:51, SEQ IDNO:61, SEQ ID NO:71, SEQ ID NO:81, SEQ ID NO:91, SEQ ID NO:101, SEQ IDNO:111, SEQ ID NO:121, SEQ ID NO:131, SEQ ID NO:141, SEQ ID NO:151, SEQID NO:161 or fragments thereof.
 14. An scfv that binds human Resistin,wherein the scfv is encoded by nucleotide sequence that is at least 90%identical to SEQ ID NO:1, SEQ ID NO:11, SEQ ID NO:21, SEQ ID NO:31, SEQID NO:41, SEQ ID NO:51, SEQ ID NO:61, SEQ ID NO:71, SEQ ID NO:81, SEQ IDNO:91, SEQ ID NO:101, SEQ ID NO:111, SEQ ID NO:121, SEQ ID NO:131, SEQID NO:141, SEQ ID NO:151, SEQ ID NO:161 or fragments thereof.
 15. Anscfv that binds human Resistin, wherein the scfv comprises SEQ ID NO:2,SEQ ID NO:12, SEQ ID NO:22, SEQ ID NO:32, SEQ ID NO:42, SEQ ID NO:52,SEQ ID NO:62, SEQ ID NO:72, SEQ ID NO:82, SEQ ID NO:92, SEQ ID NO:102,SEQ ID NO:112, SEQ ID NO:122, SEQ ID NO:132, SEQ ID NO:142, SEQ IDNO:152, SEQ ID NO:162 or fragments thereof.
 16. An scfv that binds humanResistin, wherein the scfv comprises an amino acid sequence that is atleast 90% identical to SEQ ID NO:2, SEQ ID NO:12, SEQ ID NO:22, SEQ IDNO:32, SEQ ID NO:42, SEQ ID NO:52, SEQ ID NO:62, SEQ ID NO:72, SEQ IDNO:82, SEQ ID NO:92, SEQ ID NO:102, SEQ ID NO:112, SEQ ID NO:122, SEQ IDNO:132, SEQ ID NO:142, SEQ ID NO:152, SEQ ID NO:162 or fragmentsthereof.
 17. The antibody of claim 1, further comprising a constantdomain comprising SEQ ID NO:172, SEQ ID NO:174 or a fragment thereof.18. The antibody of claim 1, further comprising a constant domain thatis at least 90% identical to SEQ ID NO:172, SEQ ID NO:174 or a fragmentthereof.
 19. A Resistin antibody comprising a heavy chain selected fromthe group consisting of SEQ ID NO:176, SEQ ID NO:180, SEQ ID NO:184, SEQID NO:188, SEQ ID NO:192, SEQ ID NO:196, SEQ ID NO:200, SEQ ID NO:204,SEQ ID NO:208, SEQ ID NO:212, SEQ ID NO:216, SEQ ID NO:220, SEQ IDNO:224, SEQ ID NO:228, SEQ ID NO:232, SEQ ID NO:236, and SEQ ID NO:240.20. A Resistin antibody comprising a light chain selected from the groupconsisting of SEQ ID NO:178, SEQ ID NO:182, SEQ ID NO:186, SEQ IDNO:190, SEQ ID NO:194, SEQ ID NO:198, SEQ ID NO:202, SEQ ID NO:206, SEQID NO:210, SEQ ID NO:214, SEQ ID NO:218, SEQ ID NO:222, SEQ ID NO:226,SEQ ID NO:230, SEQ ID NO:234, SEQ ID NO:238, and SEQ ID NO:
 242. 21. AResistin antibody comprising (a) a heavy chain selected from the groupconsisting of SEQ ID NO:176, SEQ ID NO:180, SEQ ID NO:184, SEQ IDNO:188, SEQ ID NO:192, SEQ ID NO:196, SEQ ID NO:200, SEQ ID NO:204, SEQID NO:208, SEQ ID NO:212, SEQ ID NO:216, SEQ ID NO:220, SEQ ID NO:224,SEQ ID NO:228, SEQ ID NO:232, SEQ ID NO:236, and SEQ ID NO:240 and (b) alight chain selected from the group consisting of SEQ ID NO:178, SEQ IDNO:182, SEQ ID NO:186, SEQ ID NO:190, SEQ ID NO:194, SEQ ID NO:198, SEQID NO:202, SEQ ID NO:206, SEQ ID NO:210, SEQ ID NO:214, SEQ ID NO:218,SEQ ID NO:222, SEQ ID NO:226, SEQ ID NO:230, SEQ ID NO:234, SEQ IDNO:238, and SEQ ID NO:
 242. 22. The antibody of claim 1, wherein theantibody is also cross-reactive with Resistin Like Molecule Beta(RELMβ).
 23. The scfv of claim 13, wherein the scfv is alsocross-reactive with RELMβ.
 24. A method for treating a disease, disorderor condition mediated by human Resistin in a patient comprising the stepof administering to the patient an antibody of claim
 1. 25. The methodof claim 24, wherein the disease, disorder or condition is one or moreof pulmonary hypertension, cardiac hypertrophy and failure, asthma, lunginflammation, sepsis, acute lung injury, respiratory distress syndrome,pulmonary fibrosis, scleroderma, arteriosclerosis, chronic obstructivelung disease/emphysema, normal and abnormal wound healing, cancer, cellproliferation, stem cell growth and differentiation, diabeticretinopathy, and insulin resistance.